The essential role of aggregation for the emulsifying ability of a fungal CYS-rich protein.

Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: • Two Fusarium solani strains are analyzed for their surfactant production. • A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. • An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.

Evidence of Small Fungal Cysteine-Rich Proteins Acting as Biosurfactants and Self-Assembling into Large Fibers.

Fungi produce surface-active proteins, among which hydrophobins are the most characterized and attractive also for their ability to form functional amyloids. Our most recent findings show that these abilities are shared with other classes of fungal proteins. Indeed, in this paper, we compared the characteristics of a class I hydrophobin (Vmh2 from Pleurotus ostreatus) and an unknown protein (named PAC3), extracted from the marine fungal strain Acremonium sclerotigenum, which does not belong to the same protein family based on its sequence features. They both proved to be good biosurfactants, stabilizing emulsions in several conditions (concentration, pH, and salinity) and decreasing surface tension to a comparable value to that of some synthetic surfactants. After that, we observed for both Vmh2 and PAC3 the formation of giant fibers without the need for harsh conditions or long incubation time, a remarkable ability herein reported for the first time.

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

Follicular microenvironment: Oxidative stress and adiponectin correlated with steroids hormones in women undergoing in vitro fertilization.

Research has been focused on determining the follicular microenviroment produced by the theca and granulosa cells since the molecular characterisation of this body fluid could lead to the understanding of several fertility problems. Oxidative stress may be one of the factors involved in female infertility since it plays a key role in the modulation of oocyte maturation and finally pregnancy. An increase in oxidative stress is correlated with inflammation and intense research was developed to understand the interaction between inflammation and adiponectin, based on the fact that many adipokines are inflammation related proteins linked to reactive oxygen species production. The aim of this study is to investigate the correlation between total adiponectin levels and oxidative stress amount in the serum and follicular fluid (FF) of women who undergone in vitro fertilization. Moreover we verified the expression of adiponectin in granulosa and cumulus cells. To clarify the predictive value of steroid hormones in human assisted reproduction, twelve steroid hormones in FF and serum, were quantified in a single run liquid chromatography/mass spectrometry, by using a multiple reaction monitoring mode and we related the serum and follicular fluids adiponectin levels with the concentration of the investigated steroid hormones.

Inflammation protein quantification by multiple reaction monitoring mass spectrometry in lipopolysaccharide-stimulated THP-1 cells.

RATIONALE: Inflammation is a cascade of events mediated by a cytokine network triggering the cellular response. In order to monitor the modulation of the crucial inflammatory proteins, e.g., Tumour Necrosis Factor-α (TNF-α), Interferon-γ (INF-γ), Interleukin-8 (IL-8) and Interleukin-10 (IL-10), upon stimulation with endotoxins, differentiated and undifferentiated THP-1 cells were treated with lipopolysaccharides (LPSs) from E. coli, key cell wall components of Gram-negative bacteria. METHODS: The multiple reaction monitoring mass spectrometry (MRM-MS) method was optimized by using the standard proteins to be quantified, in order to construct external calibration curves and define the analytical parameters. The developed method was used to quantify the above-mentioned inflammatory proteins in THP-1 differentiated cells upon stimulation with LPSs with high accuracy, sensitivity, and robustness. RESULTS: The analysis of such proteins in MRM mode allowed the kinetics of stimulation along the time up to 24 h to be followed and the MS results were found to be comparable with those obtained by Western-blotting. A significant increase in TNF-α release triggered a cascade mechanism leading to the production of INF-γ and IL-8. IL-10, instead, was found to be constant throughout the process. CONCLUSIONS: The developed MRM-MS method allowed the quantification of TNF-α, INF-γ, IL-8 and IL-10 along a time-course from 2 to 24 h. Hence, a trace of the kinetics of the inflammatory response in THP-1 cells upon stimulation with E. coli LPSs was obtained. Finally, the extensibility of the developed MRM method to serum samples and other matrices demonstrated the versatility of the approach and the possibility to quantify multiple target proteins in different biological samples by using a few microliters in a single analysis.

Relationship between the metabolic and lipid profile in follicular fluid of women undergoing in vitro fertilization.

Among the follicular fluid (FF) components promoting the development of the oocyte are included glycoproteins, several fatty acids, and steroid hormones synthesized by the dominant follicle. For this, the analysis of the metabolites present in FF can determine the quality of the oocyte. FF composition is in part determined by local follicular metabolic processes and in part a plasma transudate. Since the causes of impaired fertility may be due to a metabolic imbalance, metabolomics is useful to identify low molecular weight metabolites. Oxidative stress is involved in human infertility and the use of metabolomics can be crucial to identify which other metabolites besides reactive oxygen species are involved in oxidative stress correlated to infertility. To obtain new information on the study of signaling molecules in FF, the knowledge of the lipid content will be important to improve information on the understanding of follicular development. The objective of this study is to identify (a) a metabolic profile and a lipid profile of FF in women undergoing in vitro fertilization and (b) to correlate the previous information obtained regarding adiponectin and oxidative stress with the metabolic and lipid profile obtained in the present study. As result, we found an increase in oxidative stress due to both an increase of androgens and an accumulation of lipids in the follicular environment and we suggest that this might be one of the causes of reduced fertility.

Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy.

Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab (i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications.

Multiple Reaction Monitoring Tandem Mass Spectrometry Approach for the Identification of Biological Fluids at Crime Scene Investigations.

Knowledge of the nature of biofluids at a crime scene is just as important as DNA test to link the nature of the biofluid, the criminal act, and the dynamics of the crime. Identification of methods currently used for each biological fluid (blood, semen, saliva, urine) suffer from several limitations including instability of assayed biomolecules, and low selectivity and specificity; as an example of the latter issue, it is not possible to discriminate between alpha-amylase 1 (present in saliva) and alpha-amylase 2 (present in semen and vaginal secretion. In this context, the aim of the work has been to provide a predictive protein signature characteristic of each biofluid by the recognition of specific peptides unique for each protein in a single analysis. A panel of four protein biomarkers for blood, four for saliva, five for semen, and two for urine has been monitored has been monitored by using a single multiple reaction monitoring (MRM)-based method targeting concomitantly 46 different peptides. Then, The optimized method allows four biological matrices to be identified when present on their own or in 50:50 mixture with another biofluid. Finally, a valid strategy combining both DNA analysis and liquid chromatographic-tandem mass spectrometric multiple reaction monitoring (LC-MS-MRM) identification of biofluids on the same sample has been demonstrated to be particularly effective in forensic investigation of real trace evidence collected at a crime scene.

Chronic lactate exposure promotes cardiomyocyte cytoskeleton remodelling.

We investigated the effect of growing on lactate instead of glucose in human cardiomyocyte assessing their viability, cell cycle activity, oxidative stress and metabolism by a proteomic and metabolomic approach. In previous studies performed on elite players, we found that adaptation to exercise is characterized by a chronic high plasma level of lactate. Lactate is considered not only an energy source but also a signalling molecule and is referred as "lactormone"; heart is one of the major recipients of exogenous lactate. With this in mind, we used a cardiac cell line AC16 to characterize the lactate metabolic profile and investigate the metabolic flexibility of the heart. Interestingly, our data indicated that cardiomyocytes grown on lactate (72 h) show change in several proteins and metabolites linked to cell hypertrophy and cytoskeleton remodelling. The obtained results could help to understand the effect of this metabolite on heart of high-performance athletes.

Intrinsically disordered Prosystemin discloses biologically active repeat motifs.

The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.

Transcriptional and proteomic analysis of the innate immune response to microbial stimuli in a model invertebrate chordate.

Inflammatory response triggered by innate immunity can act to protect against microorganisms that behave as pathogens, with the aim to restore the homeostatic state between host and beneficial microbes. As a filter-feeder organism, the ascidian Ciona robusta is continuously exposed to external microbes that may be harmful under some conditions. In this work, we used transcriptional and proteomic approaches to investigate the inflammatory response induced by stimuli of bacterial (lipopolysaccharide -LPS- and diacylated lipopeptide - Pam2CSK4) and fungal (zymosan) origin, in Ciona juveniles at stage 4 of metamorphosis. We focused on receptors, co-interactors, transcription factors and cytokines belonging to the TLR and Dectin-1 pathways and on immune factors identified by homology approach (i.e. immunoglobulin (Ig) or C-type lectin domain containing molecules). While LPS did not induce a significant response in juvenile ascidians, Pam2CSK4 and zymosan exposure triggered the activation of specific inflammatory mechanisms. In particular, Pam2CSK4-induced inflammation was characterized by modulation of TLR and Dectin-1 pathway molecules, including receptors, transcription factors, and cytokines, while immune response to zymosan primarily involved C-type lectin receptors, co-interactors, Ig-containing molecules, and cytokines. A targeted proteomic analysis enabled to confirm transcriptional data, also highlighting a temporal delay between transcriptional induction and protein level changes. Finally, a protein-protein interaction network of Ciona immune molecules was rendered to provide a wide visualization and analysis platform of innate immunity. The in vivo inflammatory model described here reveals interconnections of innate immune pathways in specific responses to selected microbial stimuli. It also represents the starting point for studying ontogeny and regulation of inflammatory disorders in different physiological conditions.

Chronic Training Induces Metabolic and Proteomic Response in Male and Female Basketball Players: Salivary Modifications during In-Season Training Programs.

The aim of this study was to characterize the salivary proteome and metabolome of highly trained female and male young basketball players, highlighting common and different traits. A total of 20 male and female basketball players (10 female and 10 male) and 20 sedentary control subjects (10 female and 10 male) were included in the study. The athletes exercised at least five times per week for 2 h per day. Saliva samples were collected mid-season, between 9:00 and 11:00 a.m. and away from sport competition. The proteome and metabolome were analyzed by using 2DE and GC-MS techniques, respectively. A computerized 2DE gel image analysis revealed 43 spots that varied in intensity among groups. Between these spots, 10 (23.2%) were differentially expressed among male athletes and controls, 22 (51.2%) between female basketball players and controls, 11 spots (25.6%) between male and female athletes, and 13 spots (30.2%) between male and female controls. Among the proteins identified were Immunoglobulin, Alpha-Amylase, and Dermcidin, which are inflammation-related proteins. In addition, several amino acids, such as glutamic acid, lysine, ornithine, glycine, tyrosine, threonine, and valine, were increased in trained athletes. In this study, we highlight that saliva is a useful biofluid to assess athlete performance and confirm that the adaptation of men and women to exercise has some common features, but also some different sex-specific behaviors, including differential amino acid utilization and expression of inflammation-related proteins, which need to be further investigated. Moreover, in the future, it will be interesting to examine the influence of sport-type on these differences.

Multi-omic characterisation as a tool to improve knowledge, valorisation and conservation of wild fruit genetic resources: the case of Arbutus unedo L.

The valorisation and conservation of plant genetic resources (PGRs) and wild fruit PGRs are critical to ensure the maintenance of genetic and cultural heritage and to promote new perspectives on resource use. New strategies to characterize PGRs are needed, and the omics approach can provide information that is still largely unknown. The Strawberry tree (Arbutus unedo L.) is an underutilized, drought and fire-resistant species distributed in the Mediterranean area and its berries have large ethnobotanical use. Although their phenolic profile and antioxidant capacity are known, they are not well characterised, particularly from a proteomic perspective. The aim of this work is the characterisation of two ecotypes of A. unedo (Campania and Sicily) from a molecular viewpoint to valorise and encourage the preservation of this wild fruit. Samples were collected from two different geographical areas to assess whether different geographical conditions could influence the characteristics of leaves and fruits at the three stages of ripening (green, veraison, red). Proteomic analysis identified 904 proteins, of which 122 showed significance along the ripening. Some of these differentially abundant proteins, such as chalcone synthase, show a marked increase during ripening. The protein functional classes with the highest representation are involved in protein and amino acid metabolism, glycolysis and in secondary metabolism. From a proteomic perspective, there are no differences between the fruits from the two regions compared by the ripening stage. However, the pedoclimatic metabolic imprinting allowed the observation of good diversity in the metabolomic profiles between the two ecotypes, especially for anthocyanins, 4 times more abundant in the Sicilian veraisoned fruit than in the Campania one, and catechins, with double the abundance in the Campania ecotype compared to the Sicilian ecotype in the green phase, but more abundant (3x) in the Sicilian veraisoned fruit. Phenolic compounds show a 20% greater abundance in the Campania green arbutus fruit than in the Sicilian one, values that then equalise as ripening progresses. Multi-omic characterisation enhanced the knowledge on a wild fruit plant species which shows specific adaptations and responses to the environment to be considered when addressing the issue of local agrobiodiversity.

Inside out Porphyridium cruentum: Beyond the Conventional Biorefinery Concept.

Here, an unprecedented biorefinery approach has been designed to recover high-added value bioproducts starting from the culture ofPorphyridium cruentum. This unicellular marine red alga can secrete and accumulate high-value compounds that can find applications in a wide variety of industrial fields. 300 ± 67 mg/L of exopolysaccharides were obtained from cell culture medium; phycoerythrin was efficiently extracted (40% of total extract) and isolated by single chromatography, with a purity grade that allowed the crystal structure determination at 1.60 Å; a twofold increase in ß-carotene yield was obtained from the residual biomass; the final residual biomass was found to be enriched in saturated fatty acids. Thus, for the first time, a complete exploitation ofP. cruentumculture was set up.

LC-MS/MS-Based Quantification Method of Polyphenols for Valorization of Ancient Apple Cultivars from Cilento.

Safeguarding the biodiversity of plant species is of fundamental importance for their defense against pests and diseases even through the maintenance and dissemination of ancient agricultural traditions rooted within the small rural environments. The investigation area of the current research covered some municipalities belonging to the "Parco Nazionale del Cilento e Vallo di Diano" including the sub-mountainous part of "Comunità Montana del Vallo di Diano (Salerno, Campania)". Fifteen ancient apple varieties were collected from local communities to be analyzed and compared to some commercially available apples. To this aim, a Folin-Ciocâlteu assay was preliminarily used to measure the total polyphenol content in both ancient and commercial apple cultivars. Then, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis in the multiple reaction monitoring (MRM) ion mode was then implemented to detect and quantify specific polyphenols and to obtain a molecular comparison of a wide panel of polyphenols. The main finding of the present work pointed out that ancient apple cultivars are richer than commercial ones in anthocyanins, dihydrochalcones, and chlorogenic acid, whose beneficial effects on health are widely known. Thus, the safeguarding of these ancient varieties is greatly encouraged for the richness of polyphenols crucial both for the defense of plants from insects and for remarkable nutraceutical properties, in addition to the need for germplasm conservation as a source of genetic variability.

Modulation of Plasma Proteomic Profile by Regular Training in Male and Female Basketball Players: A Preliminary Study.

Monitoring fatigue and recovery during training periods contributes to identifying the best training methods to achieve sports performance. To date, little is known about sex-related differences in sports adaptations. The aim of the present study is to identify sex-related sports adaptation proteins in female basketball players and male basketball players using proteomics approach on plasma samples withdrawn from athletes during in-season training period but far from a competition. A cohort of 20 professional basketball players, 10 female (BF) and 10 male (BM), and 20 sedentary male (10 CM) and female (10 CF) as control, of comparable age and BMI, were involved in this study. Protein profiles of plasma samples obtained from BM, BF, CM, and CF were analyzed by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry. The computational 2-DE gel image analysis pointed out 33 differentially expressed protein spots (ANOVA p-value < 0.05) and differences between male and female basketball players are more evident among the players than controls. The expression profile of 54.5% of the total proteins is affected by sports activity. Furthermore, 14 proteins are differentially expressed in basket female players in comparison with their relative controls while seven are differentially expressed in basket male players in comparison with their controls. In conclusion, we identify in female athletes a reduction in proteins related to transcription regulation, most of these modulate chronic inflammation confirming the anti-inflammatory effect of regular training in female muscle metabolism. In male and female athletes, we found a decrease in Transthyretin involved in muscle homeostasis and regeneration and Dermcidin a stress-induced myokine linked to inflammatory and it will be interesting to fully understand the role of its different isoforms in male and female skeletal muscle contraction.

Oxylipin profile in saliva from patients with cystic fibrosis reveals a balance between pro-resolving and pro-inflammatory molecules.

Oxylipins are signaling molecules originated by fatty acids that modulate vascular and bronchial tone, bronchial secretion, cytokine production and immune cell activity. The unbalanced production of pro-inflammatory and pro-resolving (i.e., anti-inflammatory) oxylipins has a relevant role in the pathogenesis of pulmonary inflammation like in cystic fibrosis (CF). We analyzed by LC-MRM/MS 65 oxylipins and 4 fatty acids in resting saliva from 69 patients with CF and 50 healthy subjects (controls). The salivary levels of 48/65 oxylipins were significantly different between CF patients and controls. Among these, EpETE, DHET, 6ketoPGE1 and HDHA were significantly higher in saliva from CF patients than in controls. All these molecules display anti-inflammatory effects, i.e., releasing of bronchial and vascular tone, modulation of cytokine release. While 20-hydroxyPGF2A, PGB2, EpDPE, 9 K-12-ELA, bicyclo-PGE2, oleic acid, LTC4, linoleic acid, 15oxoEDE, 20 hydroxyPGE2 and DHK-PGD2/PGE2 (mostly associated to pro-inflammatory effects) resulted significantly lower in CF patients than in controls. Our data suggest that the salivary oxylipins profile in CF patients is addressed toward a global anti-inflammatory effect. Although these findings need be confirmed on larger populations in prospective studies, they will contribute to better understand the pathogenesis of CF chronic inflammation and to drive targeted therapies based on the modulation of oxylipins synthesis and degradation.

The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins.

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent ß-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.

An Endemic Plant of the Mediterranean Area: Phytochemical Characterization of Strawberry Tree (Arbutus unedo L.) Fruits Extracts at Different Ripening Stages.

This work focused on the extraction, quantification, and characterization of bioactive compounds of Arbutus unedo L. fruits, comparing the results obtained from the different ripening states. Extractions were performed by different methods (such as maceration extraction and ultrasonic extraction) and food grade solvents (aqueous and hydroalcoholic solvents) in each of the all ripening states (four states considered, associated with four different colors, i.e., green, yellow, orange, and red). The presence of (poly)phenols was quantified and characterized, and scavenging activity was determined by the Folin-Ciocâlteu reagent and the DPPH method, respectively. The content of bioactive compounds was characterized by LC-MS/MS, such as multiple reaction monitoring (MRM) mass spectrometry. The results showed that ultrasound-assisted extraction (UAE) performed better than maceration extraction; ethanol-water mixture extracts showed a more positive effect than the use of aqueous extracts regarding the content of total phenolic compounds. Overall, the total phenolic compounds in the EtOH:H2O mixture at a ratio of 7:3 (v:v) were higher than that of the other solvents for both extraction methods. Some bioactive molecules were characterized for the first time in the extracts of A. unedo. The chemical profile of the strawberry tree extracts depended on the degree of fruit ripeness. The results suggest that A. unedo fruits may be of great interest for food and nutraceutical applications.

Association of the serological status of rheumatoid arthritis patients with two circulating protein biomarkers: A useful tool for precision medicine strategies.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and presence of systemic autoantibodies, with a great clinical and molecular heterogeneity. Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are routinely used for the diagnosis of RA. However, additional serological markers are needed to improve the clinical management of this disease, allowing for better patient stratification and the desirable application of precision medicine strategies. In the present study, we investigated those systemic molecular changes that are associated with the RF and ACPA status of RA patients. To achieve this objective, we followed a proteomic biomarker pipeline from the discovery phase to validation. First, we performed an iTRAQ-based quantitative proteomic experiment on serum samples from the RA cohort of the Hospital of Santiago de Compostela (CHUS). In this discovery phase, serum samples from the CHUS cohort were pooled according to their RF/ACPA status. Shotgun analysis revealed that, in comparison with the double negative group (RF-/ACPA-), the abundance of 12 proteins was altered in the RF+/ACPA+ pool, 16 in the RF+/ACPA- pool and 10 in the RF-/ACPA+ pool. Vitamin D binding protein and haptoglobin were the unique proteins increased in all the comparisons. For the verification phase, 80 samples from the same cohort were analyzed individually. To this end, we developed a Multiple Reaction Monitoring (MRM) method that was employed in a comprehensive targeted analysis with the aim of verifying the results obtained in the discovery phase. Thirty-one peptides belonging to 12 proteins associated with RF and/or ACPA status were quantified by MRM. In a final validation phase, the serum levels of alpha-1-acid glycoprotein 1 (A1AG1), haptoglobin (HPT) and retinol-binding protein 4 (RET4) were measured by immunoassays in the RA cohort of the Hospital of A Coruña (HUAC). The increase of two of these putative biomarkers in the double seropositive group was validated in 260 patients from this cohort (p = 0.009 A1AG1; p = 0.003 HPT). The increased level of A1AG1 showed association with RF rather than ACPA (p = 0.023), whereas HPT showed association with ACPA rather than RF (p = 0.013). Altogether, this study has allowed a further classification of the RA seropositive patients into two novel clusters: RF+A1AG+ and ACPA+HPT+. The determination of A1AG1 and HPT in serum would provide novel information useful for RA patient stratification, which could facilitate the effective implementation of personalized medicine in routine clinical practice.